Thermostable DNA polymerase from a hyperthermophilic archaeon strain KOD1

ABSTRACT

A nucleic acid amplifying enzyme having a short reaction time and high fidelity is provided. The enzyme of this invention is a thermostable DNA polymerase having a nucleic acid extension rate of at least 30 bases per second and a 3&#39;-5&#39; exonuclease activity. Also provided are a method and kit for amplifying nucleic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 08/656,005 filedMay 24, 1996, which is incorporated herein in its entirety by referencethereto.

FIELD OF THE INVENTION

The present invention relates to a method of amplifying nucleic acidwherein DNA or RNA is amplified within a short reaction time and with ahigh fidelity, to a method of identifying nucleic acid utilizing saidamplifying method and to a DNA polymerase and a reagent kit used forthose methods.

[Prior Art]

Many studies have been made already for DNA polymerase of mesophilicmicroorganism such as Escherichia coli and for DNA polymerase derivedfrom phages infectable by the mesophilic microorganisms. In addition,many studies have been also made already for heat stable DNA polymeraseswhich are useful in a recombinant DNA technique by means of nucleic acidamplification such as a polymerase chain reaction (PCR). Examples of theheat-stable polymerases which are used for the PCR are DNA polymerase(Tth polymerase) mostly derived from Thermus thermophilus and DNApolymerase (Taq polymerase) derived from Thermus aguaticus. Other knownexamples are DNA polymerase (Pfu polymerase) derived from Pyrococcusfuriosus and DNA polymerase (Vent polymerase) derived from Thermococcuslitoralis.

[Problems to be Solved by the Invention]

However, with the Taq polymerase, fidelity and thermostability upon thesynthesis of DNA are not sufficient. Although the Pfu polymeraseexhibiting excellent fidelity and thermostability has been developed,said Pfu polymerase has some problems that its DNA extension rate isslow and a processivity is low whereby it has been used only for aspecific PCR.

Recently, a PCR whereby 20 kb or more DNA is amplified (hereinafter,referred to as a long-PCR) has been developed. In said long-PCR, bothTaq polymerase and Pfu polymerase are mixed whereby properties of bothenzymes are utilized.

However, when two enzymes having different properties are used in thesame reaction system, some discrepancies might occur in theirappropriate reaction conditions whereby there is a question whether thehigh extension rate and fidelity which are the advantages of each ofthose enzymes can be still maintained. Moreover, because of thedifference in the thermostabilities and in the composition of the stocksolutions of both enzymes, there is a question as to the stability whenthey are stored in the same container.

In view of the above, there has been a keen demand for novelthermostable polymerase which exhibits both of those advantages.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a stained alkaline agarose gel on which DNA extended byHyperthermophilic Archaeon Strain KOD1 polymerase for various lengths oftime was separated.

FIG. 2a is a stained alkaline agarose gel on which DNA extended forvarious times using the present polymerase and using Pfu polymerase wasseparated.

FIG. 2b is a stained alkaline agarose gel on which DNA extended by DeepVent polymerase and DNA extended by Taq polymerase was separated.

FIG. 3 is a comparison of PCR products obtained using the presentpolymerase at different reaction times.

FIG. 4 is a schematic drawing of the construction of a recombinantvector encoding the present polymerase gene.

FIG. 5 is a stained SDS-PAGE gel showing the molecular weight of thepresent polymerase.

FIG. 6 is a stained SDS-PAGE gel showing the PCR products of a reactiondriven by the present polymerase.

FIG. 7 is a schematic drawing of the intron and exon structure of theHyperthermophilic Archaeon strain KOD1 polymerase gene.

SUMMARY OF THE INVENTION

The present inventors have succeeded in preparing a thermostable DNApolymerase from a hyperthermophilic archaeon strain KOD1, and, when itsproperties are investigated, it has been found that said DNA polymeraseexhibits the advantages of the above-mentioned two enzymes, i.e. highextension rate and high fidelity, whereby the present invention has beenachieved.

Thus, the present invention relates to a method for amplifying a targetnucleic acid comprising reacting the target nucleic acid with four kindsof dNTP and primer complementary to said target nucleic acid in a buffersolution which contains a thermostable DNA polymerase having a DNAextension rate of at least 30 bases/second and a 3'-5' exonucleaseactivity such that the above mentioned primer is annealed to the targetnucleic acid and an extention product is synthesized from the primer.

The present invention further relates to a method for amplifying atarget nucleic acid in a sample wherein each target nucleic acidconsists of two separate complementary strands which comprises thefollowing steps A to D, characterized in that a thermostable DNApolymerase having a DNA extension rate of at least 30 bases/second and a3'-5' exonuclease activity is used as a thermostable DNA polymerase;

A: modifying the target nucleic acid, if necessary, to producesingle-stranded nucleic acids;

B: reacting the single-stranded nucleic acids with four kinds of dNTPand primers, wherein said primers are selected so as to be sufficientlycomplementary to different strands of target nucleic acid to annealtherewith, in a buffer solution which contains a thermostable DNApolymerase such that the above mentioned primers are annealed to thesingle-stranded nucleic acids and extention products are synthesizedfrom the primers,

C: separating the primer extention products from the templates on whichthey are synthesized to produce single-stranded nucleic acids; and

D: repeatedly conducting the above mentioned steps B and C.

The present invention further relates to a method for detecting a targetnucleic acid in a sample wherein each target nucleic acid consists oftwo separate complementary strands which comprises the following steps Ato E, characterized in that a thermostable DNA polymerase having a DNAextension rate of at least 30 bases/second and a 3'-5' exonucleaseactivity is used as a thermostable DNA polymerase;

A: modifying the target nucleic acid, if necessary, to producesingle-stranded nucleic acids;

B: reacting the single-stranded nucleic acids with four kinds of dNTPand primers, wherein said primers are selected so as to be sufficientlycomplementary to different strands of target nucleic acid to annealtherewith, in a buffer solution which contains a thermostable DNApolymerase such that the above mentioned primers are annealed to thesingle-stranded nucleic acids and extention products are synthesizedfrom the primers,

C: separating the primer extention products from the templates on whichthey are synthesized to produce single-stranded nucleic acids;

D: repeatedly conducting the above mentioned steps B and C, and

E: detecting an amplified nucleic acid.

The present invention further relates to a reagent kit for amplifyingtarget nucleic acid which comprises primers, wherein said primers areselected so as to be sufficiently complementary to different strands oftarget nucleic acid to anneal therewith, four kinds of dNTP, divalentcation, thermostable DNA polymerase having a DNA extension rate of atleast 30 bases/second and a 3'-5' exonuclease activity and buffersolution.

The present invention further relates to a reagent kit for detectingtarget nucleic acid which comprises primers, wherein said primers areselected so as to be sufficiently complementary to different strands oftarget nucleic acid to anneal therewith, four kinds of dNTP, divalentcation, thermostable DNA polymerase having a DNA extension rate of atleast 30 bases/second and a 3'-5' exonuclease activity, amplifyingbuffer solution, a probe capable of hybridizing with amplified nucleicacid and a detection buffer solution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a thermostable DNA polymerase which isobtainable from a strain KOD1 which belongs to a hyperthermophilicarchaeon strain.

The present invention relates to an isolated DNA comprising a nucleotidesequence that encodes the thermostable DNA polymerase derived from aKOD1 strain which belongs to hyperthermophilic archaeon.

The present invention further relates to a recombinant DNA expressionvector that comprises the DNA sequence inserted into a vector, whereinthe DNA sequence encodes the thermostable DNA polymerase derived from aKOD1 strain which belongs to hyperthermophilic archaeon.

The present invention further relates to a transformed recombinant hostcell using a recombinant DNA expression vector that comprises the DNAsequence inserted into a vector, wherein the DNA sequence encodes thethermostable DNA polymerase derived from a KOD1 strain which belongs tohyperthermophilic archaeon.

The present invention relates to a method for producing a DNA polymeraseobtainable from a KOD1 strain which belongs to hyperthermophilicarchaeon, comprising culturing recombinant host cells which aretransformed by a recombinant DNA expression vector that comprises theDNA sequence inserted into a vector, wherein the DNA sequence encodesthe thermostable DNA polymerase derived from a KOD1 strain which belongsto hyperthermophilic archaeon, and recovering the produced thermostableDNA polymerase.

The present invention further relates to a method for purifying the DNApolymerase obtainable from a KOD1 strain which belongs tohyperthermophilic archaeon, comprising culturing the recombinant hostcells which are transformed by a recombinant DNA expression vector thatcomprises the DNA sequence inserted into a vector, wherein the DNAsequence encodes the thermostable DNA polymerase derived from a KOD1strain which belongs to hyperthermophilic archaeon, and further (a)recovering the cultured recombinant host cells, lysing them andpreparing the cell extract, and (b) removing the impurified proteinsderived from recombinant host cells.

The nucleic acid which is to be amplified by the present invention isDNA or RNA. There is no restriction at all for the sample in which sucha nucleic acid is contained.

The thermostable enzyme which is used in the present invention is athermostable DNA polymerase having at least 30 bases/second of DNAextension rate and having a 3'-5' exonuclease activity. Its specificexample is a DNA polymerase derived from a hyperthermophilic archaeonstrain KOD1 (called a KOD polymerase) and said enzyme may be either athermostable enzyme purified from nature or an enzyme manufactured by agene recombination technique.

The DNA extension rate in the present invention is calculated from therelationship between the reaction time and the size of the synthesizedDNA in the reaction of various kinds of DNA polymerases such as ROD,Pfu, Deep Vent, Taq, etc. (5U) in each buffer using a substrate preparedby annealing a single-stranded DNA (1.6 μg) of M13 with a primer (16pmoles) complementary thereto. It is essential in the present inventionthat the DNA extension rate is at least 30 bases/second.

The DNA extension rates for each of the polymerases are 105-130bases/second for ROD polymerase, 24.8 bases/second for Pfu polymerase,23.3 bases/second for Deep Vent polymerase and 61.0 bases/second for Taqpolymerase.

On the other hand, it is essential in the present invention that thethermostable DNA polymerase has a 3'-5' exonuclease activity.

In the present invention, the 3'-5' exonuclease activity is determinedby checking the rate of release of ³ H under the optimum condition foreach polymerase using a substrate wherein the 3'-end of the lambda-DNAdigested with HindIII labeled with [³ H]TTP.

In the 3'-5' exonuclease activity of each polymerase, free-³ H is foundto be only 10-20%. In the case of Taq polymerase and Tth polymeraseafter an incubation period of three hours, in KOD polymerase and Pfupolymerase, it is 50-70%.

It has been confirmed that the KOD polymerase used in the presentinvention has a 3'-5' exonuclease activity and that, in the gene whichcodes for KOD polymerase, there is a DNA conserved sequence showing a3'-5' exonuclease activity, which is the same as in the case of Pfupolymerase.

In the present invention, the fact whether there is a 3'-5' exonucleaseactivity is checked in such a manner that KOD polymerase is allowed tostand, using a DNA fragment into which the DNA of [³H]TTP-labelled-lambda-DNA digested with HindIII is incorporated as asubstrate, at the reaction temperature of 75° C. in a buffer (20 mMTris-HCl of pH 6.5, 10 mM KCl, 6 mM (NH₄)₂ SO₄, 2 mM MgCl₂, 0.1% TritonX-100 and 10 μg/ml BSA) and the ratio of the free-[³ H]TTP isdetermined.

At the same time, Taq polymerase and Tth polymerase having no 3'-5'exonuclease activity and Pfu polymerase having a 3'-5' exonucleaseactivity were checked using a buffer for each of them by the same manneras in the control experiments. The titer of each of the used polymeraseswas made 2.5 units.

The substrate DNA was prepared in such a manner that, first, 0.2 mM ofdATP, dGTP, dCTP and [³ H]TTP were added to 10 μg of lambda-DNA digestedwith HindIII, the 3'-end was elongated by Klenow polymerase, then DNAfragments were recovered by extracting with phenol and precipitated withethanol and free mononucleotides were removed by a Spin column(manufactured by Clontech).

In the case of KOD polymerase and Pfu polymerase, 50-70% of free [³H]TTP were detected after an incubation period of three hours, in thecase of Taq polymerase and Tth polymerase, only 10-20% of free [³ H]TTPwas noted.

It is preferred that said thermostable DNA polymerase contains an aminoacid sequence given in SEQ ID No. 1.

It is also preferred that said thermostable DNA polymerase is an enzymehaving the following physical and chemical properties.

Action: It has a DNA synthetic activity and a 3'-5' exonucleaseactivity.

DNA extension rate: at least 30 bases/second

Optimum pH: 6.5-7.5 (at 75° C.)

Optimum temperature: 75° C.

Molecular weight: about 88-90 Kda

Amino acid sequence: as mentioned in SEQ ID No. 1

An example of the methods for manufacturing DNA polymerase derived froma hyperthermophilic archaeon strain KOD1 is that thermostable DNApolymerase gene was cloned from strain KOD1 which was isolated from asolfatara at a wharf on Kodakara Island, Kagoshima so that a recombinantexpression vector was constructed, then a transformant prepared bytransformation by said recombinant vector was cultured and thethermostable DNA polymerase was collected from the culture followed bypurifying.

In the present invention, the DNA polymerase derived from theabove-mentioned hyperthermophilic archaeon strain KOD1 has a DNAsynthesizing activity and a 3'-5' exonuclease activity and has a DNAextension rate of at least 30 bases/second. This property is used forconducting an amplification of nucleic acid.

The amplifying method of the present invention includes the followingsteps A to D.

A: modifying the target nucleic acid, if necessary, to producesingle-stranded nucleic acids;

B: reacting the single-stranded nucleic acids with four kinds of dNTPand primers, wherein said primers are selected so as to be sufficientlycomplementary to different strands of target nucleic acid to annealtherewith, in a buffer solution which contains a thermostable DNApolymerase such that the above mentioned primers are annealed to thesingle-stranded nucleic acids and extention products are synthesizedfrom the primers,

C: separating the primer extention products from the templates on whichthey are synthesized to produce single-stranded nucleic acids; and

D: repeatedly conducting the above mentioned steps B and C.

In the step A, the target nucleic acid is denatured if necessary to givea single-stranded nucleic acid. The means therefor may be a thermaltreatment, a chemical denaturation or an enzymatic treatment.Preferably, it is a thermal treatment.

In the step B, said single-stranded nucleic acid is made to react withfour kinds of dNTP (dATP, dGTP, dCTP and dTTP or dUTP) and primers withregular and inverted directions having complementary base sequences tothe target nucleic acid in a buffer solution containing a thermostableDNA polymerase so that said primers are annealed to the single-strandednucleic acid to conduct a primer extention reaction.

A primer with a regular direction and that with an inverted directionhaving complementary base sequences to the target nucleic acid areoligonucleotides having a base sequence which is complementary to onestrand of target nucleic acid and is homologous to the other strand.Accordingly, one primer may be complementary to another primer elongate.

Preferred buffer solutions containing a thermostable DNA polymerase areTris buffers containing divalent cation such as magnesium ion.

An example of the conditions for conducting an elongation reaction byannealing the primer is a method in which a cycle of 98° C./1 second-1minute and 68° C./1 second-10 minutes is repeated for 30 times.

The step of separating an elongated primer for making a single strand inthe step C may be a thermal treatment, a chemical treatment or anenzymatic treatment. Preferably, it is a thermal treatment or anenzymatic treatment using RNase.

In the step D, the above-mentioned steps B and C are repeated. To bemore specific, it is preferred that heating and cooling of 98° C./20seconds and 68° C./30 seconds are repeated at least for 30 cycles.

An amplifying method of the present invention is applicable to a PCR foramplifying a DNA of 20 kb or more (hereinafter, referred to as along-PCR) as well. In this long-PCR, advantages of both high DNAextension rate of Taq polymerase and high fidelity in DNA synthesiscaused by a 3'-5' exonuclease activity of Pfu polymerase are necessaryand both enzymes are used after mixing them. In this case, there is aquestion on a stability when both enzymes are stored in the samecontainer because of the difference between their thermostabilities andthat between the compositions of their stored solutions. However, in theDNA polymerase derived from a hyperthermophilic archaeon strain KOD1, asingle enzyme exhibits both high DNA extension rate and high fidelitydue to its 3',-5' exonuclease activity whereby it is possible that along-PCR can be conducted by its sole use.

In the present invention, the amplified product produced by theabove-mentioned amplification such as a labeled probe is used whereby atarget nucleic acid can be detected.

Labeled probe is an oligonucleotide having a base sequence which iscomplementary to a target nucleic acid and is bonded with a labeledsubstance or a labeled binding substance.

Examples of the labeled substance are enzymes such as alkalinephosphatase, peroxidase and galactosidase, fluorescent substances andradioactive substances while examples of the labeled binding substancesare biotin and digoxigenin. Labeled substance may be bonded via biotin,digoxigenin or avidin.

A method of introducing those labels into a probe is that, during thesynthesis of oligonucleotide, dNTP to which those labeled substances orlabeled binding substances are bonded is used as one of the componentsof dNTP whereby a synthesis is conducted.

Examples of detecting a nucleic acid bonded with a labeled probe areconventionally known methods such as a Southern hybridization and aNorthern hybridization. In those methods, the fact that a hybrid isformed when single-stranded DNA and RNA are complementary each other isutilized whereby unknown nucleic acid fraction group is subjected to anagarose electrophoresis to separate it by size, then the nucleic acidfraction in the gel is subjected, for example, to an alkali treatment,the resulting single strand is transferred to a filter, immobilized andhybridized with a labeled probe.

As to a detection of the label in case an alkaline phosphatase is usedas a labeled substance, when a chemoluminescent substrate such as a1,2-dioxetane compound (PPD) is made to react therewith, only nucleicacid forming a hybrid is illuminated. This is sensitized to an X-rayfilm whereby the size of the target nucleic acid and its position onelectrophoresis can be confirmed.

A reagent kit for nucleic acid amplification according to the presentinvention contains primers of regular and inverted directions havingbase sequences complementary to target nucleic acid, four kinds of dNTP,divalent cation, thermostable DNA polymerase having a DNA extension rateof at least 30 bases/second and having a 3'-5' exonuclease activity anda buffer solution.

An example of divalent cation is magnesium ion. Its concentration ispreferably about 1-3 mM. Examples of the buffer solution are tris buffer(pH 6.5, 75° C.) and tricine buffer (pH 6.5, 75° C.).

A specific example of the composition is as follows.

20 mM Tris-HCl (pH 6.5, 75° C.)

10 mM KCl

6 mM (NH₄)₂ SO₄

1-3 mM MgCl₂

0.1% Triton X-100

10 μg/ml BSA

20-200 μM dNTPs

0.1 pM-1 μM primer

0.1-250 ng template DNA.

A reagent kit for nucleic acid amplification according to the presentinvention contains a nucleic acid amplifying reagent comprising primersof regular and inverted directions having base sequences complementaryto target nucleic acid, four kinds of dNTP, divalent cation,thermostable DNA polymerase having a DNA extension rate of at least 30bases/second and having a 3'-5' exonuclease activity and a buffersolution for amplification, a target nucleic acid probe and a buffer fordetection. The buffer for detection varies depending upon the label. Forexample, it includes a color reagent or a luminous reagent.

KOD1 which is a kind of hyperthermophilic archaeon used in the presentinvention is a strain isolated from a solfatara at a wharf on KodakaraIsland, Kagoshima.

Mycological properties of said strain are as follows.

Shape of cells: coccus, diplococcus; having flagella.

Temperature range for the growth: 65-100° C.

Optimum temperature for the growth: 95° C.

pH range for the growth: 5-9

Optimum pH: 6

Optimum salt concentration: 2-3%

Auxotrophy: heterotrophic

Oxygen demand: aerophobic

Cell membrane lipids: ether type

GC content of DNA: 38%

The hyperthermophilic archaeon strain KOD1 was a coccus having adiameter of about 1 μm and had plural polar flagella. From themycological properties of the strain, its close relationship with PfuDNA polymerase-productive bacterium (Pyrococcus furiosus) and with Tli(Vent) DNA polymerase-productive bacterium (Thermococcus litoralis) wassuggested.

Cloning of the thermostable DNA polymerase gene of the present inventionis carried out as follows.

Thus, the cloning method is that a primer is designed and synthesizeddepending upon an amino acid sequence in a conserved region of Pfu DNApolymerase (Nucleic Acids Research, 1993, vol.21, No. 2, 259-265).

First, a PCR is conducted using the above-prepared primers (e.g., SEQ IDNos. 7 and 8) taking chromosomal DNA of the hyperthermophilic archaeonstrain KOD1 as a template to amplify the DNA fragment. The DNA sequence(e.g., SEQ ID No. 9) of the amplified fragment is determined and, afterconfirming that the originally set amino acid sequence is coded for, aSouthern hybridization is conducted to the cleaved product of thechromosomal DNA with a restriction enzyme using said fragment as aprobe. It is preferred that the approximate size of the fragmentcontaining the target DNA polymerase gene is limited to about 4-7 Kbp.

Then DNA fragment of about 4-7 Kbp is recovered from the gel, a DNAlibrary is prepared by Escherichia coli using said fragment and a colonyhybridization is carried out using the above-mentioned PCR-amplified DNAfragment (e.g., SEQ ID No. 9) to collect a clone strain.

The DNA polymerase gene of the strain KOD1 cloned in the presentinvention is composed of 5010 bases (estimated numbers of amino acids:1670) (SEQ ID No. 5).

Upon comparison with other DNA polymerases, there is a conserved regionof αDNA polymerase which is an eukaryote type (Regions 1-5) in the geneof the present invention. In addition, there are EXO 1,2,3 which are3'→5' exonuclease motif at the N terminal of said gene. In the conservedregions (Regions 1, 2) of the thermostable DNA polymerase gene derivedfrom the hyperthermophilic archaeon strain KOD1, each of the interveningsequences is present and they are connected in a form where the openreading frame (ORF) is conserved.

When the thermostable DNA polymerase gene of the hyperthermophilicarchaeon strain KOD1 is compared with Pfu DNA polymerase gene derivedfrom Pyrococcus furiosus (Japanese Laid-Open Patent PublicationHei-05/328969) and with Tli (Vent) DNA polymerase gene derived fromThermococcus litoralis (Japanese Laid-Open Patent PublicationHe-06/7160) which are known enzymes, intervening sequence is present inthe gene of the strain KOD1 of the present invention while there is nointervening sequence in the gene of the above-mentioned Pfu DNApolymerase and, in the Tli DNA polymerase gene, there are two kinds ofintervening sequences but they are present within Regions 2 and 3 whichare conserved regions and that greatly differs from the location wherethe intervening sequence in the thermostable DNA polymerase gene of KOD1strain of the present invention exists (Refer to FIG. 7).

The gene of the present invention is a DNA which codes for the DNApolymerase derived from the hyperthermophilic archaeon strain KOD1. Anexample of said DNA contains a base sequence which codes for the aminoacid sequence mentioned in SEQ ID No. 1 or 5. Further, such a DNAcontains a base sequence mentioned in SEQ ID No. 5 or 6 or a partthereof.

In order to express the thermostable DNA polymerase derived from thehyperthermophilic archaeon strain KOD1 of the present invention inEscherichia coli, the intervening sequences of 1374-2453 bp and2708-4316 bp in the base sequence shown by SEQ ID No. 5 are removed bymeans of a PCR gene fusion to construct a DNA polymerase gene of acomplete form. To be specific, a PCR is conducted on a cloned genecontaining the intervening sequence by a combination of three pairs ofprimers to amplify the three fragments which are divided by theintervening sequence. In designing the primers used here, a part of thefragment which is to be bonded to its terminal is contained in its5'-end. Then a PCR is conducted using the fragments to be bondedutilizing the duplicated sequence of the terminal whereby each of thefragments is bonded. Further PCR is conducted by the same manner usingthe resulting two kinds of fragments to give a DNA polymerase gene in acomplete form containing no DNA polymerase gene derived from the strainKOD1 containing no intervening sequence.

Any vector may be used in the present invention so far as it makescloning and expression of the thermostable DNA polymerase derived fromKOD1 possible and its example is phage and plasmid. An example of theplasmid is a plasmid vector wherein an expression induced by T7 promoteris possible such as pET-8c. Other examples of the plasmid are pUC19,pBR322, pBluescript, pSP73, pGW7, pET3A and pET11C and so on. Examplesof the phage are lambda gt11, lambda DASH and lambda ZapII and so on.

Examples of the host cell used in the present invention are Escherichiacoli and yeasts. Examples of Escherichia coli are JM109, 101, XL1, PR1and BL21(DE3)pysS and so on.

In the present invention, the gene coding for the thermostable DNApolymerase derived from the above-mentioned KOD1 is inserted into theabove-mentioned vector to give a recombinant vector and the host cell issubjected to a transformation using said recombinant vector.

In the production method of the present invention, the above-mentionedrecombinant host cell is cultured whereby the thermostable DNApolymerase gene derived from the strain KOD1 is induced and expressed.The culture medium used for the culture of the recombinant host cell andthe condition therefor follow the conventional methods.

In a specific example, Escherichia coli which is transformed by pET-8cplasmid containing a DNA polymerase gene in a complete form containingno intervening sequence derived from the strain KOD1 is cultured, forexample, in a TB medium whereby an induction treatment is conducted. Itis preferred that the induction treatment of T7 promoter is carried outby addition of isopropyl-thio-β-D-galactoside.

The purifying method of the present invention includes, after culturingthe recombinant host cells, a step wherein (a) recombinant host cellsare collected, lysed and the cell extract is prepared and a step wherein(b) impure protein derived from the host cells is removed.

The thermostable DNA polymerase which is produced from the recombinanthost cells is separated and recovered from the culture liquid by meansof centrifugation or the like after culturing the host bacterial cellsin a medium followed by inducing. After said bacterial cells areresuspended in a buffer, they are lysed by means of ultrasonictreatment, Dyno mill, French press, etc. Then a thermal treatment isconducted and the heat stable DNA polymerase is recovered from thesupernatant fluid. In disintegrating the bacterial cells, ultrasonictreatment, Dyno mill and French press method are preferred.

A thermal treatment is preferred as one of the steps for removing theimpure protein derived from the host cells. The condition for thethermal treatment is at 70° C. or higher or, preferably, at 90° orhigher. Other means for removing the impure protein are variouschromatographic techniques.

Molecular weight of the thermostable DNA polymerase derived from thehyperthermophilic archaeon strain KOD1 obtained as such is about 90 KDa(cf. FIG. 5).

When a polymerase chain reaction is conducted using said thermostableDNA polymerase, a sufficient amplification of the aimed DNA fragments isconfirmed (cf. FIG. 6).

Now the present invention will be illustrated by referring partly to thedrawings wherein:

FIG. 1 is a photographic picture of electrophoresis as a substitute fora drawing and shows the result of the measurement of the DNA extensionrate of the KOD polymerase;

FIG. 2 is a photographic picture of electrophoresis as a substitute fora drawing and shows the comparison of the DNA extension rate of variousthermostable DNA polymerases in which FIG. 2a shows the cases of KODpolymerase and Pfu polymerase while FIG. 2b shows the cases of Deep Ventpolymerase and Taq polymerase;

FIG. 3 is a photographic picture of electrophoresis as a substitute fora drawing and shows the comparison of the PCR due to the difference inthe reaction time of various thermostable DNA polymerase;

FIG. 4 shows the constructive charts of the recombinant expressionvector;

FIG. 5 is a photographic picture of electrophoresis as a substitute fora drawing and shows the result of the measurement of molecular weight ofthe thermostable DNA polymerase derived from KOD1;

FIG. 6 is a photographic picture of electrophoresis as a substitute fora drawing and shows the result of the PCR by the thermostable DNApolymerase derived from KOD1; and

FIG. 7 is drawings which shows a comparison of the DNA polymerase genederived from the hyperthermophilic archaeon strain KOD1 with thethermostable DNA polymerase gene derived from Pyrococcus furiosus andthat derived from Thermococcus litoralis which are thought to be similarbacteria.

EXAMPLE 1 Cloning of DNA Polymerase Gene Derived from hyperthermophilicarchaeon strain KOD1

The hyperthermophilic archaeon strain KOD1 isolated in Kodakara Island,Kagoshima was cultured at 95° C. and then the bacterial cells wererecovered. Chromosomal DNA of the hyperthermophilic archaeon strain KOD1was prepared by a conventional method from the resulting bacterialcells.

Two kinds of primers (5'-GGATTAGTATAGTGCCAATGGAAGGCGAC-3' [SEQ ID No. 7]and 5'-GAGGGCGAAGTTTATTCCGAGCTT-3' [SEQ ID No. 8]) were synthesizedbased upon the amino acid sequence at the conserved region of the DNApolymerase (Pfu polymerase) derived from Pyrococcus furiosus. A PCR wascarried out using those two primers where the prepared chromosomal DNAwas used as a template.

After the base sequence (SEQ ID No. 9) of the PCR-amplified DNA fragmentwas determined and the amino acid sequence (SEQ ID No. 10) wasdetermined, a Southern hybridization was conducted using said amplifiedDNA fragment to the product of the strain KOD1 chromosomal DNA treatedwith a restriction enzyme whereby the size of the fragment coding forthe DNA polymerase was calculated (about 4-7 Kbp). Further, the DNAfragment of this size was recovered from agarose gel, inserted into aplasmid pBS (manufactured by Stratgene) and Escherichia coli (E. coli JM109) was transformed by this mixture to prepare a library.

A colony hybridization was conducted using a probe (SEQ ID No. 9) usedfor the Southern hybridization to obtain a clone strain (E. coliJM109/pBSKOD1) which is thought to contain the DNA polymerase genederived from strain KOD1.

EXAMPLE 2 Determination of Base Sequence of the Clone Fragment

A plasmid pBSKOD1 was recovered from the clone strain E. coliJM109/pBSKOD1 obtained in Example 1 and its base sequence (SEQ ID No. 5)was determined by a conventional method. Further, the amino acidsequence was presumed from the determined base sequence. The DNApolymerase gene derived from KOD1 strain comprised 5010 bases wherein1670 amino acids were coded.

EXAMPLE 3 Construction of Recombinant Expression Vector

In order to prepare a complete polymerase gene, the intervening sequenceparts at two places (1374-2453 bp and 2708-4316 bp) were removed by aPCR fusion method. In the PCR fusion method, three pairs of primers (SEQID Nos.11-16) were combined using a primer recovered from the clonestrain as a template and a PCR was conducted for each of them to amplifythree fragments wherefrom the intervening sequences were removed. Atthat time, the primer used for the PCR was designed in such a mannerthat the side which binds to another fragment has the same sequence asthe binding partner has. In addition, a design was conducted in such amanner that different restriction enzyme sites (EcoRV at N-terminalwhile BamHI at C-terminal) were created at both ends.

After that, among the PCR-amplified fragments, that which is located atthe central part of the structure and that which is located at theN-terminal side are mixed and a PCR was conducted using each of thefragments as a primer. At the same time, the fragment located at thecentral part of the structure and that located at the C-terminal sideare mixed and a PCR was conducted using each of the fragments as aprimer. Two kinds of fragments obtained as such were subjected to a PCRonce again to give gene fragments in a complete form having nointervening sequence, having EcoRV and BamHI sites at the N- andC-terminals, respectively and coding for the DNA polymerase derived fromstrain KOD1.

Further, said gene was subcloned using an expression vector which can beinduced by T7 promoter, an NcoI/BamHI site of pET-8c and thepreviously-created restriction enzyme site to give a recombinantexpression vector (pET-pol).

EXAMPLE 4 Expression and Purification of DNA Polymerase Derived fromKOD1

Escherichia coli (BL21(DE3)) was transformed using a recombinantexpression vector (pET-pol) obtained in Example 3, the resultingtransformant was cultured in a TB medium (mentioned in MolecularCloning, p.A.2, 1989) and, at one hour before collecting the bacterialcells, an induction treatment of T7 promoter was conducted by additionof isopropylthio-β-D-galactopyrenoside. Bacterial cells were recoveredfrom the cultured liquid by means of centrifugation. They wereresuspended in a buffer and disintegrated by an ultrasonic treatment togive a cell extract. In order to remove the impure protein derived fromthe host cells, the disintegrated cell solution was treated at 94° C.for 20 minutes whereby the impure protein derived from the host cellstrifugation to give a thermostable. DNA polymerase derived from strainKOD1.

The Escherichia coli BL21 (DE3) pER-pol was deposited on Apr. 22, 1996under the Budepest Treaty at National Institute of Bioscience andHuman-Technology Agency of Industrial Science and Technology (1-3,Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, JAPAN) in accordance withthe Budapest Treaty under the accession number FERM BP-5513.

EXAMPLE 5 Purification of Thermostable DNA Polymerase Derived from KOD1

Molecular weight of the thermostable DNA polymerase derived from KOD1obtained in Example 4 was calculated by means of an SDS-PAGE methodwhereby it was found to be about 86-92 kDa (FIG. 5). Further, a PCR wasconducted using the thermostable DNA polymerase derived from KOD1obtained in Example 4 and the known template primer whereupon a DNAfragment which was to be a target was confirmed (FIG. 6) by the samemanner as in the case where the thermostable DNA polymerase derived fromThermococcus litoralis was used and a high thermostable DNA polymeraseactivity was confirmed.

COMPARATIVE EXAMPLE 1 Comparison with the Thermostable DNA PolymeraseGene Derived from Pyrococcus furiosus or from Thermococcus litoraliswhich are to be Similar to the Hyperthermophilic archaeon strain KOD1 ofthe Present Invention

Amino acid sequences were estimated from the DNA sequences of the DNApolymerase gene derived from the hyperthermophilic archaeon strain KOD1of the present invention (SEQ ID No. 6), the thermostable DNA polymerasegene derived from Pyrococcus furiosus (Japanese Laid-Open PatentPublication Hei-5/328969) and the thermostable DNA polymerase genederived from Thermococcus litoralis (Japanese Laid-Open PatentPublication Hei-6/7160) and were compared and investigated.

In the DNA polymerase derived from KOD1 of the present invention, therewere Regions 1-5 which were the conserved regions of αDNA polymerase ofan eurokaryotic type. Further, there were EXO1, 2 and 3 which were 3'→5'exonuclease motifs at the N-terminal side. However, in each of theRegion 1 and Region 2 which were the αDNA polymerase conserved regions,there were intervening sequences IVS-A and IVS-B (refer to FIG. 7).

On the other hand, in Pfu polymerase which is a thermostable DNApolymerase derived from Pyrococcus furiosus, there was no interveningsequence. In the case of Vent polymerase which is a thermostable DNApolymerase derived from Thermococcus litoralis, there were theintervening sequences (IVS1 and IVS2) in the αDNA polymerase conservedregions (Region 2 and Region 3) (refer to FIG. 7).

EXAMPLE 6 Measurement of DNA Extension Rate of the DNA PolymeraseDerived from Hyperthermophilic archaeon strain KOD1

DNA prepared by annealing the M13mp18DNA with M13P7 primer having a basesequence as mentioned in SEQ ID No. 2 was used as a substrate and therate of synthesizing the DNA in a reaction buffer solution [20 mMTris-HCl (pH 7.5 at 75° C.), 10 mM KCl, 6 mM (NH₄)₂ SO₄, 2 mM MgCl₂,0.1% Triton X-100 and 10 μg/ml nuclease-free BSA] containing the DNApolymerase derived from the hyperthermophilic archaeon strain KOD1manufactured in Examples 1-5 was investigated for the reaction time of20, 40, 60, 80 and 100 seconds (FIG. 1) or 40, 60, 80 and 100 seconds(FIG. 2). The results are given in FIG. 1 and in FIG. 2.

A part of the DNA sample during the elongation reaction was taken outfor each reaction time and was added to a reaction stopping solution (60mM EDTA, 60 μM NaOH, 0.1% BPB and 30% glycerol) in the same amount.

The DNA samples obtained in the above process were separated andanalyzed by means of an alkaline agarose electrophoresis and the size ofthe synthesized DNA was checked.

1, 2, 3, 4 and 5 in FIG. 1 show the results of the reactions for 0.3minute (20 seconds), 0.7 minute (40 seconds), 1 minute (60 seconds), 1.3minutes (80 seconds) and 1.7 minutes (100 seconds), respectively. It isapparent from FIG. 1 that the DNA extension rate of the DNA polymerasederived from the hyperthermophilic archaeon strain KOD1 was 105bases/second.

1, 2, 3 and 4 in FIG. 2 show the results of the reaction for 0.7 minute(40 seconds), 1 minute (60 seconds), 1.3 minutes (80 seconds) and 1.7minutes (100 seconds), respectively. It is apparent from FIG. 2 that theDNA extension rate of the DNA polymerase derived from thehyperthermophilic archaeon strain KOD1 was 138 bases/second.

On the other hand, the DNA synthesizing rate of each of Pfu polymerase(Stratgene), Deep Vent polymerase (New England Biolabo) and Taqpolymerase (Takara Shuzo) was measured by the same manner in each of thebuffers therefor (FIG. 2a and FIG. 2b). The DNA extension rates of thoseDNA polymerases were 24.8 bases/second for Pfu polymerase, 23.2bases/second for Deep Vent polymerase and 61.0 bases/second for Taqpolymerase.

From the above results, it was suggested that the DNA extension rate ofthe DNA polymerase derived from the hyperthermophilic archaeon strainKOD1 was about six-fold of those of Pfu polymerase and Deep Ventpolymerase and about two-fold of that of Taq polymerase.

EXAMPLE 7 Measurement of Fidelity of the DNA Polymerase Derived from theHyperthermophilic archaeon strain KOD1 in the Reaction for the Synthesisof DNA

A rate for resulting in an error in the DNA synthesis was measured by amethod of Kunkel (Kunkel, 1985, Journal of Biological Chemistry, 260,5787-5796). In this method, a DNA synthesis reaction was conducted usinga DNA polymerase derived from the hyperthermophilic archaeon strain KOD1manufactured in Examples 1-5 using an M13mp18DNA having a gap at a lacZpart containing a part of the genes coding for β-galactosidase as asubstrate and transfected to E. coli JM109 in an NZY medium containing5-bromo-4-chloro-3-indolyl-β-D-galactoside andisopropyl-thio-β-D-galactoside using an M13mp18DNA in which lacZ partwas double-stranded.

When β-galactosidase wherein a function is lost or lowered was expresseddue to a reading error or a frame shift during the synthetic reaction ofDNA, it is not possible to utilize5-bromo-4-chloro-3-indolyl-β-D-galactoside whereupon the color of plaguebecomes colorless or light blue. On the other hand, when there is noerror in the synthesized DNA and a complete β-galactosidase wasexpressed, plaque becomes blue. The rate of induction of error wasmeasured in the DNA synthesis from the rate of the sum of colorless andlight blue plaque to the total plaque.

The rate of induction of error in the DNA synthesis was also measuredfor Pfu polymerase (Stratgene), Taq polymerase (Takara Shuzo) and deltaTth polymerase (Toyobo) which were made to react by the same manner.

Further, the rate of induction of error in the DNA synthesis was alsomeasured for a mixture of Taq polymerase and Pfu polymerase. The resultsare given in Table 1.

                  TABLE 1                                                         ______________________________________                                        Measurement of Fidelity in the Reaction of DNA Synthesis of                   DNA Polymerase Derived from Hyperthermophilic archaeon strain KODl                                                 Mutant                                   Enzyme    Light Blue                                                                              White  Mutant                                                                             Total                                                                               Frequence (10.sup.-4)                   ______________________________________                                        KODl pol.                                                                              12       11      23    6619 37.7                                     Pfu               15                                                                                  15                                                                                   30                                                                               7691                                                                                     39.0                             Taq               30                                                                                  24                                                                                   54                                                                               4141                                                                                    130                               ▴Tth                                                                        70         45                                                                                  115                                                                               7375                                                                                    156                               Taq/Pfu(20:1)                                                                          10              20                                                                                  30                                                                               4238                                                                                     63.7                             Taq/Pfu(50:1)                                                                          10              13                                                                                  23                                                                               4489                                                                                     53.5                             ______________________________________                                    

It is apparent from Table 1 that the fidelity of the DNA polymerasederived from hyperthermophilic archaeon strain KOD1 in the DNA synthesisreaction is suggested to be superior to Taq polymerase and same as Pfupolymerase. In addition, a mixture of Taq polymerase and Pfu polymeraseexhibits a medium fidelity that it is superior to Taq polymerase andinferior to Pfu polymerase.

EXAMPLE 8 Comparison in PCR of Various Thermostable DNA Polymerases bythe Difference in the Reaction Time

Lambda-DNA (3 μg) was used as a target nucleic acid; oligonucleotideshaving a sequence as mentioned in SEQ ID Nos. 3 and 4 were used asprimers; and a buffer containing 20 mM Tri-HCl (pH 7.5 at 75° C.), 10 mMKCl, 6 mM (NH₄)₂ SO₄, 2 mM MgCl₂, 0.1% Triton X-100, 10 μg/ml BSA and200 μM dNTPs was used as a buffer. DNA polymerase derived fromhyperthermophilic archaeon strain KOD1 (KOD polymerase), Taq polymerasewhich is widely used for PCR and Pfu polymerase which exhibits 3'-5'exonuclease activity were also used as the thermostable DNA polymerases.The used titer of each polymerase was 2 units.

A PCR amplification reaction was conducted using a DNA Thermal Cycler(Perkin-Elmer) in a schedule wherein a cycle comprising 94° C./20seconds and 68° C./x second (x: reaction time) was repeated for 30times. In the case of the DNA polymerase derived from thehyperthermophilic archaeon strain KOD1 (KOD polymerase), amplificationof the target DNA was confirmed by conducting 30 cycles of 94° C./20seconds-68° C./1 second while, in the case of Taq polymerase,amplification of DNA was first confirmed by conducting 30 cycles of 94°C./20 seconds-68° C./10 seconds. In the case of Pfu polymerase,amplification of DNA was at least confirmed by conducting 30 cycles of94° C./20 seconds-68° C./1 minute. The results are given in FIG. 3.

In the present invention, it is possible to amplify the DNA with a highfidelity within a short reaction time when a DNA polymerase derived fromhyperthermophilic archaeon strain KOD1 which is a thermostable DNApolymerase having at least 30 bases/second of DNA extension rate andhaving a 3'-5' exonuclease activity. When this method is made into aform of a kit, it is possible to improve the simplicity and convenience.In addition, when only one kind of thermostable DNA polymerase havingboth high extension rate (at least 30 bases/second) which has not beenavailable yet and 3'-5' exonuclease activity is used, it is possible toshorten the time for the primer extention reaction and to amplify therelatively big product with a high fidelity.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 16                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 774 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu As - #p Gly Lys Pro Val Ile         #15                                                                           - Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Ly - #s Ile Glu Tyr Asp Arg         #             30                                                              - Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Ly - #s Asp Asp Ser Ala Ile         #         45                                                                  - Glu Glu Val Lys Lys Ile Thr Ala Glu Arg Hi - #s Gly Thr Val Val Thr         #     60                                                                      - Val Lys Arg Val Glu Lys Val Gln Lys Lys Ph - #e Leu Gly Arg Pro Val         # 80                                                                          - Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gl - #n Asp Val Pro Ala Ile         #                 95                                                          - Arg Asp Lys Ile Arg Glu His Gly Ala Val Il - #e Asp Ile Tyr Glu Tyr         #           110                                                               - Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile As - #p Lys Gly Leu Val Pro         #       125                                                                   - Met Glu Gly Asp Glu Glu Leu Lys Met Leu Al - #a Phe Asp Ile Gln Thr         #   140                                                                       - Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gl - #y Pro Ile Leu Met Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Il - #e Thr Trp Lys Asn Val         #               175                                                           - Asp Leu Pro Tyr Val Asp Val Val Ser Thr Gl - #u Arg Glu Met Ile Lys         #           190                                                               - Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pr - #o Asp Val Leu Ile Thr         #       205                                                                   - Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Le - #u Lys Lys Arg Cys Glu         #   220                                                                       - Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg As - #p Gly Ser Glu Pro Lys         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Gln Arg Met Gly Asp Arg Phe Ala Val Gl - #u Val Lys Gly Arg Ile         #               255                                                           - His Phe Asp Leu Tyr Pro Val Ile Arg Arg Th - #r Ile Asn Leu Pro Thr         #           270                                                               - Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Ph - #e Gly Gln Pro Lys Glu         #       285                                                                   - Lys Val Tyr Ala Glu Glu Ile Thr Pro Ala Tr - #p Glu Thr Gly Glu Asn         #   300                                                                       - Leu Glu Arg Val Ala Arg Tyr Ser Met Glu As - #p Ala Lys Val Thr Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Al - #a Gln Leu Ser Arg Leu         #               335                                                           - Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Se - #r Ser Thr Gly Asn Leu         #           350                                                               - Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Gl - #u Arg Asn Glu Leu Ala         #       365                                                                   - Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Ar - #g Arg Arg Gln Ser Tyr         #   380                                                                       - Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gl - #y Leu Trp Glu Asn Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Se - #r Ile Ile Ile Thr His         #               415                                                           - Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gl - #y Cys Lys Glu Tyr Asp         #           430                                                               - Val Ala Pro Gln Val Gly His Arg Phe Cys Ly - #s Asp Phe Pro Gly Phe         #       445                                                                   - Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Gl - #u Arg Gln Lys Ile Lys         #   460                                                                       - Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Gl - #u Arg Lys Leu Leu Asp         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala As - #n Ser Tyr Tyr Gly Tyr         #               495                                                           - Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Ly - #s Glu Cys Ala Glu Ser         #           510                                                               - Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Me - #t Thr Ile Lys Glu Ile         #       525                                                                   - Glu Glu Lys Tyr Gly Phe Lys Val Ile Tyr Se - #r Asp Thr Asp Gly Phe         #   540                                                                       - Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Th - #r Val Lys Lys Lys Ala         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Met Glu Phe Leu Asn Tyr Ile Asn Ala Lys Le - #u Pro Gly Ala Leu Glu         #               575                                                           - Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Ph - #e Phe Val Thr Lys Lys         #           590                                                               - Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Il - #e Thr Thr Arg Gly Leu         #       605                                                                   - Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Al - #a Lys Glu Thr Gln Ala         #   620                                                                       - Arg Val Leu Glu Ala Leu Leu Lys Asp Gly As - #p Val Glu Lys Ala Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Arg Ile Val Lys Glu Val Thr Glu Lys Leu Se - #r Lys Tyr Glu Val Pro         #               655                                                           - Pro Glu Lys Leu Val Ile His Glu Gln Ile Th - #r Arg Asp Leu Lys Asp         #           670                                                               - Tyr Lys Ala Thr Gly Pro His Val Ala Val Al - #a Lys Arg Leu Ala Ala         #       685                                                                   - Arg Gly Val Lys Ile Arg Pro Gly Thr Val Il - #e Ser Tyr Ile Val Leu         #   700                                                                       - Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Il - #e Pro Phe Asp Glu Phe         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Ty - #r Tyr Ile Glu Asn Gln         #               735                                                           - Val Leu Pro Ala Val Glu Arg Ile Leu Arg Al - #a Phe Gly Tyr Arg Lys         #           750                                                               - Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Va - #l Gly Leu Ser Ala Trp         #       765                                                                   - Leu Lys Pro Lys Gly Thr                                                         770                                                                       - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24GTCA CGAC                                                  - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               GGTT                                                       - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #                24CGGT CAAT                                                  - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 5342 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE: Hyperthermophilic arch - #aeon                    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TG - #CCGGTTTT         60                                                                          - ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TA - #CAATAAAG        120                                                                          #GAC ACT GAC       173T TATGGGGGAT GAAAG ATG ATC CTC                          #   Met Ile Leu Asp Thr Asp                                                   #  5                                                                          - TAC ATA ACC GAG GAT GGA AAG CCT GTC ATA AG - #A ATT TTC AAG AAG GAA          221                                                                          Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Ar - #g Ile Phe Lys Lys Glu           #             20                                                              - AAC GGC GAG TTT AAG ATT GAG TAC GAC CGG AC - #T TTT GAA CCC TAC TTC          269                                                                          Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Th - #r Phe Glu Pro Tyr Phe           #         35                                                                  - TAC GCC CTC CTG AAG GAC GAT TCT GCC ATT GA - #G GAS GTC AAG AAG ATA          317                                                                          Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Gl - #u Glu Val Lys Lys Ile           #     50                                                                      - ACC GCC GAG AGG CAC GGG ACG GTT GTA ACG GT - #T AAG CGG GTT GAA AAG          365                                                                          Thr Ala Glu Arg His Gly Thr Val Val Thr Va - #l Lys Arg Val Glu Lys           # 70                                                                          - GTT CAG AAG AAG TTC CTC GGG AGA CCA GTT GA - #G GTC TGG AAA CTC TAC          413                                                                          Val Gln Lys Lys Phe Leu Gly Arg Pro Val Gl - #u Val Trp Lys Leu Tyr           #                 85                                                          - TTT ACT CAT CCG CAG GAC GTC CCA GCG ATA AG - #G GAC AAG ATA CGA GAG          461                                                                          Phe Thr His Pro Gln Asp Val Pro Ala Ile Ar - #g Asp Lys Ile Arg Glu           #            100                                                              - CAT GGA GCA GTT ATT GAC ATC TAC GAG TAC GA - #C ATA CCC TTC GCC AAG          509                                                                          His Gly Ala Val Ile Asp Ile Tyr Glu Tyr As - #p Ile Pro Phe Ala Lys           #       115                                                                   - CGC TAC CTC ATA GAC AAG GGA TTA GTG CCA AT - #G GAA GGC GAC GAG GAG          557                                                                          Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro Me - #t Glu Gly Asp Glu Glu           #   130                                                                       - CTG AAA ATG CTC GCC TTC GAC ATT CAA ACT CT - #C TAC CAT GAG GGC GAG          605                                                                          Leu Lys Met Leu Ala Phe Asp Ile Gln Thr Le - #u Tyr His Glu Gly Glu           135                 1 - #40                 1 - #45                 1 -       #50                                                                           - GAG TTC GCC GAG GGG CCA ATC CTT ATG ATA AG - #C TAC GCC GAC GAG GAA          653                                                                          Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Se - #r Tyr Ala Asp Glu Glu           #               165                                                           - GGG GCC AGG GTG ATA ACT TGG AAG AAC GTG GA - #T CTC CCC TAC GTT GAC          701                                                                          Gly Ala Arg Val Ile Thr Trp Lys Asn Val As - #p Leu Pro Tyr Val Asp           #           180                                                               - GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG CG - #C TTC CTC CGT GTT GTG          749                                                                          Val Val Ser Thr Glu Arg Glu Met Ile Lys Ar - #g Phe Leu Arg Val Val           #       195                                                                   - AAG GAG AAA GAC CCG GAC GTT CTC ATA ACC TA - #C AAC GGC GAC AAC TTC          797                                                                          Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Ty - #r Asn Gly Asp Asn Phe           #   210                                                                       - GAC TTC GCC TAT CTG AAA AAG CGC TGT GAA AA - #G CTC GGA ATA AAC TTC          845                                                                          Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu Ly - #s Leu Gly Ile Asn Phe           215                 2 - #20                 2 - #25                 2 -       #30                                                                           - GCC CTC GGA AGG GAT GGA AGC GAG CCG AAG AT - #T CAG AGG ATG GGC GAC          893                                                                          Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys Il - #e Gln Arg Met Gly Asp           #               245                                                           - AGG TTT GCC GTC GAA GTG AAG GGA CGG ATA CA - #C TTC GAT CTC TAT CCT          941                                                                          Arg Phe Ala Val Glu Val Lys Gly Arg Ile Hi - #s Phe Asp Leu Tyr Pro           #           260                                                               - GTG ATA AGA CGG ACG ATA AAC CTG CCC ACA TA - #C ACG CTT GAG GCC GTT          989                                                                          Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Ty - #r Thr Leu Glu Ala Val           #       275                                                                   - TAT GAA GCC GTC TTC GGT CAG CCG AAG GAG AA - #G GTT TAC GCT GAG GAA         1037                                                                          Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu Ly - #s Val Tyr Ala Glu Glu           #   290                                                                       - ATA ACA CCA GCC TGG GAA ACC GGC GAG AAC CT - #T GAG AGA GTC GCC CGC         1085                                                                          Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn Le - #u Glu Arg Val Ala Arg           295                 3 - #00                 3 - #05                 3 -       #10                                                                           - TAC TCG ATG GAA GAT GCG AAG GTC ACA TAC GA - #G CTT GGG AAG GAG TTC         1133                                                                          Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Gl - #u Leu Gly Lys Glu Phe           #               325                                                           - CTT CCG ATG GAG GCC CAG CTT TCT CGC TTA AT - #C GGC CAG TCC CTC TGG         1181                                                                          Leu Pro Met Glu Ala Gln Leu Ser Arg Leu Il - #e Gly Gln Ser Leu Trp           #           340                                                               - GAC GTC TCC CGC TCC AGC ACT GGC AAC CTC GT - #T GAG TGG TTC CTC CTC         1229                                                                          Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Va - #l Glu Trp Phe Leu Leu           #       355                                                                   - AGG AAG GCC TAT GAG AGG AAT GAG CTG GCC CC - #G AAC AAG CCC GAT GAA         1277                                                                          Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pr - #o Asn Lys Pro Asp Glu           #   370                                                                       - AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT GA - #A GGA GGC TAT GTA AAA         1325                                                                          Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr Gl - #u Gly Gly Tyr Val Lys           375                 3 - #80                 3 - #85                 3 -       #90                                                                           - GAG CCC GAG AGA GGG TTG TGG GAG ACC ATA GT - #G TAC CTA GAT TTT AGA         1373                                                                          Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Va - #l Tyr Leu Asp Phe Arg           #               405                                                           - TGC CAT CCA GCC GAT ACG AAG GTT GTC GTC AA - #G GGG AAG GGG ATT ATA         1421                                                                          Cys His Pro Ala Asp Thr Lys Val Val Val Ly - #s Gly Lys Gly Ile Ile           #           420                                                               - AAC ATC AGC GAG GTT CAG GAA GGT GAC TAT GT - #C CTT GGG ATT GAC GGC         1469                                                                          Asn Ile Ser Glu Val Gln Glu Gly Asp Tyr Va - #l Leu Gly Ile Asp Gly           #       435                                                                   - TGG CAG AGA GTT AGA AAA GTA TGG GAA TAC GA - #C TAC AAA GGG GAG CTT         1517                                                                          Trp Gln Arg Val Arg Lys Val Trp Glu Tyr As - #p Tyr Lys Gly Glu Leu           #   450                                                                       - GTA AAC ATA AAC GGG TTA AAG TGT ACG CCC AA - #T CAT AAG CTT CCC GTT         1565                                                                          Val Asn Ile Asn Gly Leu Lys Cys Thr Pro As - #n His Lys Leu Pro Val           455                 4 - #60                 4 - #65                 4 -       #70                                                                           - GTT ACA AAG AAC GAA CGA CAA ACG AGA ATA AG - #A GAC AGT CTT GCT AAG         1613                                                                          Val Thr Lys Asn Glu Arg Gln Thr Arg Ile Ar - #g Asp Ser Leu Ala Lys           #               485                                                           - TCT TTC CTT ACT AAA AAA GTT AAG GGC AAG AT - #A ATA ACC ACT CCC CTT         1661                                                                          Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Il - #e Ile Thr Thr Pro Leu           #           500                                                               - TTC TAT GAA ATA GGC AGA GCG ACA AGT GAG AA - #T ATT CCA GAA GAA GAG         1709                                                                          Phe Tyr Glu Ile Gly Arg Ala Thr Ser Glu As - #n Ile Pro Glu Glu Glu           #       515                                                                   - GTT CTC AAG GGA GAG CTC GCT GGC ATA CTA TT - #G GCT GAA GGA ACG CTC         1757                                                                          Val Leu Lys Gly Glu Leu Ala Gly Ile Leu Le - #u Ala Glu Gly Thr Leu           #   530                                                                       - TTG AGG AAA GAC GTT GAA TAC TTT GAT TCA TC - #C CGC AAA AAA CGG AGG         1805                                                                          Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Se - #r Arg Lys Lys Arg Arg           535                 5 - #40                 5 - #45                 5 -       #50                                                                           - ATT TCA CAC CAG TAT CGT GTT GAG ATA ACC AT - #T GGG AAA GAC GAG GAG         1853                                                                          Ile Ser His Gln Tyr Arg Val Glu Ile Thr Il - #e Gly Lys Asp Glu Glu           #               565                                                           - GAG TTT AGG GAT CGT ATC ACA TAC ATT TTT GA - #G CGT TTG TTT GGG ATT         1901                                                                          Glu Phe Arg Asp Arg Ile Thr Tyr Ile Phe Gl - #u Arg Leu Phe Gly Ile           #           580                                                               - ACT CCA AGC ATC TCG GAG AAG AAA GGA ACT AA - #C GCA GTA ACA CTC AAA         1949                                                                          Thr Pro Ser Ile Ser Glu Lys Lys Gly Thr As - #n Ala Val Thr Leu Lys           #       595                                                                   - GTT GCG AAG AAG AAT GTT TAT CTT AAA GTC AA - #G GAA ATT ATG GAC AAC         1997                                                                          Val Ala Lys Lys Asn Val Tyr Leu Lys Val Ly - #s Glu Ile Met Asp Asn           #   610                                                                       - ATA GAG TCC CTA CAT GCC CCC TCG GTT CTC AG - #G GGA TTC TTC GAA GGC         2045                                                                          Ile Glu Ser Leu His Ala Pro Ser Val Leu Ar - #g Gly Phe Phe Glu Gly           615                 6 - #20                 6 - #25                 6 -       #30                                                                           - GAC GGT TCA GTA AAC AGG GTT AGG AGG AGT AT - #T GTT GCA ACC CAG GGT         2093                                                                          Asp Gly Ser Val Asn Arg Val Arg Arg Ser Il - #e Val Ala Thr Gln Gly           #               645                                                           - ACA AAG AAC GAG TGG AAG ATT AAA CTG GTG TC - #A AAA CTG CTC TCC CAG         2141                                                                          Thr Lys Asn Glu Trp Lys Ile Lys Leu Val Se - #r Lys Leu Leu Ser Gln           #           660                                                               - CTT GGT ATC CCT CAT CAA ACG TAC ACG TAT CA - #G TAT CAG GAA AAT GGG         2189                                                                          Leu Gly Ile Pro His Gln Thr Tyr Thr Tyr Gl - #n Tyr Gln Glu Asn Gly           #       675                                                                   - AAA GAT CGG AGC AGG TAT ATA CTG GAG ATA AC - #T GGA AAG GAC GGA TTG         2237                                                                          Lys Asp Arg Ser Arg Tyr Ile Leu Glu Ile Th - #r Gly Lys Asp Gly Leu           #   690                                                                       - ATA CTG TTC CAA ACA CTC ATT GGA TTC ATC AG - #T GAA AGA AAG AAC GCT         2285                                                                          Ile Leu Phe Gln Thr Leu Ile Gly Phe Ile Se - #r Glu Arg Lys Asn Ala           695                 7 - #00                 7 - #05                 7 -       #10                                                                           - CTG CTT AAT AAG GCA ATA TCT CAG AGG GAA AT - #G AAC AAC TTG GAA AAC         2333                                                                          Leu Leu Asn Lys Ala Ile Ser Gln Arg Glu Me - #t Asn Asn Leu Glu Asn           #               725                                                           - AAT GGA TTT TAC AGG CTC AGT GAA TTC AAT GT - #C AGC ACG GAA TAC TAT         2381                                                                          Asn Gly Phe Tyr Arg Leu Ser Glu Phe Asn Va - #l Ser Thr Glu Tyr Tyr           #           740                                                               - GAG GGC AAG GTC TAT GAC TTA ACT CTT GAA GG - #A ACT CCC TAC TAC TTT         2429                                                                          Glu Gly Lys Val Tyr Asp Leu Thr Leu Glu Gl - #y Thr Pro Tyr Tyr Phe           #       755                                                                   - GCC AAT GGC ATA TTG ACC CAT AAC TCC CTG TA - #C CCC TCA ATC ATC ATC         2477                                                                          Ala Asn Gly Ile Leu Thr His Asn Ser Leu Ty - #r Pro Ser Ile Ile Ile           #   770                                                                       - ACC CAC AAC GTC TCG CCG GAT ACG CTC AAC AG - #A GAA GGA TGC AAG GAA         2525                                                                          Thr His Asn Val Ser Pro Asp Thr Leu Asn Ar - #g Glu Gly Cys Lys Glu           775                 7 - #80                 7 - #85                 7 -       #90                                                                           - TAT GAC GTT GCC CCA CAG GTC GGC CAC CGC TT - #C TGC AAG GAC TTC CCA         2573                                                                          Tyr Asp Val Ala Pro Gln Val Gly His Arg Ph - #e Cys Lys Asp Phe Pro           #               805                                                           - GGA TTT ATC CCG AGC CTG CTT GGA GAC CTC CT - #A GAG GAG AGG CAG AAG         2621                                                                          Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Le - #u Glu Glu Arg Gln Lys           #           820                                                               - ATA AAG AAG AAG ATG AAG GCC ACG ATT GAC CC - #G ATC GAG AGG AAG CTC         2669                                                                          Ile Lys Lys Lys Met Lys Ala Thr Ile Asp Pr - #o Ile Glu Arg Lys Leu           #       835                                                                   - CTC GAT TAC AGG CAG AGG GCC ATC AAG ATC CT - #G GCA AAC AGC ATC CTA         2717                                                                          Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Le - #u Ala Asn Ser Ile Leu           #   850                                                                       - CCC GAG GAA TGG CTT CCA GTC CTC GAG GAA GG - #G GAG GTT CAC TTC GTC         2765                                                                          Pro Glu Glu Trp Leu Pro Val Leu Glu Glu Gl - #y Glu Val His Phe Val           855                 8 - #60                 8 - #65                 8 -       #70                                                                           - AGG ATT GGA GAG CTC ATA GAC CGG ATG ATG GA - #G GAA AAT GCT GGG AAA         2813                                                                          Arg Ile Gly Glu Leu Ile Asp Arg Met Met Gl - #u Glu Asn Ala Gly Lys           #               885                                                           - GTA AAG AGA GAG GGC GAG ACG GAA GTG CTT GA - #G GTC AGT GGG CTT GAA         2861                                                                          Val Lys Arg Glu Gly Glu Thr Glu Val Leu Gl - #u Val Ser Gly Leu Glu           #           900                                                               - GTC CCG TCC TTT AAC AGG AGA ACT AAC AAG GC - #C GAG CTC AAG AGA GTA         2909                                                                          Val Pro Ser Phe Asn Arg Arg Thr Asn Lys Al - #a Glu Leu Lys Arg Val           #       915                                                                   - AAG GCC CTG ATT AGG CAC GAT TAT TCT GGC AA - #G GTC TAC ACC ATC AGA         2957                                                                          Lys Ala Leu Ile Arg His Asp Tyr Ser Gly Ly - #s Val Tyr Thr Ile Arg           #   930                                                                       - CTG AAG TCG GGG AGG AGA ATA AAG ATA ACC TC - #T GGC CAC AGC CTC TTC         3005                                                                          Leu Lys Ser Gly Arg Arg Ile Lys Ile Thr Se - #r Gly His Ser Leu Phe           935                 9 - #40                 9 - #45                 9 -       #50                                                                           - TCT GTG AGA AAC GGG GAG CTC GTT GAA GTT AC - #G GGC GAT GAA CTA AAT         3053                                                                          Ser Val Arg Asn Gly Glu Leu Val Glu Val Th - #r Gly Asp Glu Leu Lys           #               965                                                           - CCA GGT GAC CTC GTT GCA GTC CCG CGG AGA TT - #G GAG CTT CCT GAG AGA         3101                                                                          Pro Gly Asp Leu Val Ala Val Pro Arg Arg Le - #u Glu Leu Pro Glu Arg           #           980                                                               - AAC CAC GTG CTG AAC CTC GTT GAA CTG CTC CT - #T GGA ACG CCA GAA GAA         3149                                                                          Asn His Val Leu Asn Leu Val Glu Leu Leu Le - #u Gly Thr Pro Glu Glu           #       995                                                                   - GAA ACT TTG GAC ATC GTC ATG ACG ATC CCA GT - #C AAG GGT AAG AAG AAC         3197                                                                          Glu Thr Leu Asp Ile Val Met Thr Ile Pro Va - #l Lys Gly Lys Lys Asn           #  10105                                                                      - TTC TTT AAA GGG ATG CTC AGG ACT TTG CGC TG - #G ATT TTC GGA GAG GAA         3245                                                                          Phe Phe Lys Gly Met Leu Arg Thr Leu Arg Tr - #p Ile Phe Gly Glu Glu           #               10301020 - #                1025                              - AAG AGG CCC AGA ACC GCG AGA CGC TAT CTC AG - #G CAC CTT GAG GAT CTG         3293                                                                          Lys Arg Pro Arg Thr Ala Arg Arg Tyr Leu Ar - #g His Leu Glu Asp Leu           #              10450                                                          - GGC TAT GTC CGG CTT AAG AAG ATC GGC TAC GA - #A GTC CTC GAC TGG GAC         3341                                                                          Gly Tyr Val Arg Leu Lys Lys Ile Gly Tyr Gl - #u Val Leu Asp Trp Asp           #          10605                                                              - TCA CTT AAG AAC TAC AGA AGG CTC TAC GAG GC - #G CTT GTC GAG AAC GTC         3389                                                                          Ser Leu Lys Asn Tyr Arg Arg Leu Tyr Glu Al - #a Leu Val Glu Asn Val           #      10750                                                                  - AGA TAC AAC GGC AAC AAG AGG GAG TAC CTC GT - #T GAA TTC AAT TCC ATC         3437                                                                          Arg Tyr Asn Gly Asn Lys Arg Glu Tyr Leu Va - #l Glu Phe Asn Ser Ile           #  10905                                                                      - CGG GAT GCA GTT GGC ATA ATG CCC CTA AAA GA - #G CTG AAG GAG TGG AAG         3485                                                                          Arg Asp Ala Val Gly Ile Met Pro Leu Lys Gl - #u Leu Lys Glu Trp Lys           #               11101100 - #                1105                              - ATC GGC ACG CTG AAC GGC TTC AGA ATG AGA AA - #G CTC ATT GAA GTG GAC         3533                                                                          Ile Gly Thr Leu Asn Gly Phe Arg Met Arg Ly - #s Leu Ile Glu Val Asp           #              11250                                                          - GAG TCG TTA GCA AAG CTC CTC GGC TAC TAC GT - #G AGC GAG GGC TAT GCA         3581                                                                          Glu Ser Leu Ala Lys Leu Leu Gly Tyr Tyr Va - #l Ser Glu Gly Tyr Ala           #          11405                                                              - AGA AAG CAG AGG AAT CCC AAA AAC GGC TGG AG - #C TAC AGC GTG AAG CTC         3629                                                                          Arg Lys Gln Arg Asn Pro Lys Asn Gly Trp Se - #r Tyr Ser Val Lys Leu           #      11550                                                                  - TAC AAC GAA GAC CCT GAA GTG CTG GAC GAT AT - #G GAG AGA CTC GCC AGC         3677                                                                          Tyr Asn Glu Asp Pro Glu Val Leu Asp Asp Me - #t Glu Arg Leu Ala Ser           #  11705                                                                      - AGG TTT TTC GGG AAG GTG AGG CGG GGC AGG AA - #C TAC GTT GAG ATA CCG         3725                                                                          Arg Phe Phe Gly Lys Val Arg Arg Gly Arg As - #n Tyr Val Glu Ile Pro           #               11901180 - #                1185                              - AAG AAG ATC GGC TAC CTG CTC TTT GAG AAC AT - #G TGC GGT GTC CTA GCG         3773                                                                          Lys Lys Ile Gly Tyr Leu Leu Phe Glu Asn Me - #t Cys Gly Val Leu Ala           #              12050                                                          - GAG AAC AAG AGG ATT CCC GAG TTC GTC TTC AC - #G TCC CCG AAA GGG GTT         3821                                                                          Glu Asn Lys Arg Ile Pro Glu Phe Val Phe Th - #r Ser Pro Lys Gly Val           #          12205                                                              - CGG CTG GCC TTC CTT GAG GGG TAC TCA TCG GC - #G ATG GCG ACG TCC ACC         3869                                                                          Arg Leu Ala Phe Leu Glu Gly Tyr Ser Ser Al - #a Met Ala Thr Ser Thr           #      12350                                                                  - GAA CAA GAG ACT CAG GCT CTC AAC GAA AAG CG - #A GCT TTA GCG AAC CAG         3917                                                                          Glu Gln Glu Thr Gln Ala Leu Asn Glu Lys Ar - #g Ala Leu Ala Asn Gln           #  12505                                                                      - CTC GTC CTC CTC TTG AAC TCG GTG GGG GTC TC - #T GCT GTA AAA CTT GGG         3965                                                                          Leu Val Leu Leu Leu Asn Ser Val Gly Val Se - #r Ala Val Lys Leu Gly           #               12701260 - #                1265                              - CAC GAC AGC GGC GTT TAC AGG GTC TAT ATA AA - #C GAG GAG CTC CCG TTC         4013                                                                          His Asp Ser Gly Val Tyr Arg Val Tyr Ile As - #n Glu Glu Leu Pro Phe           #              12850                                                          - GTA AAG CTG GAC AAG AAA AAG AAC GCC TAC TA - #C TCA CAC GTG ATC CCC         4061                                                                          Val Lys Leu Asp Lys Lys Lys Asn Ala Tyr Ty - #r Ser His Val Ile Pro           #          13005                                                              - AAG GAA GTC CTG AGC GAG GTC TTT GGG AAG GT - #T TTC CAG AAA AAC GTC         4109                                                                          Lys Glu Val Leu Ser Glu Val Phe Gly Lys Va - #l Phe Gln Lys Asn Val           #      13150                                                                  - AGT CCT CAG ACC TTC AGG AAG ATG GTC GAG GA - #C GGA AGA CTC GAT CCC         4157                                                                          Ser Pro Gln Thr Phe Arg Lys Met Val Glu As - #p Gly Arg Leu Asp Pro           #  13305                                                                      - GAA AAG GCC CAG AGG CTC TCC TGG CTC ATT GA - #G GGG GAC GTA GTG CTC         4205                                                                          Glu Lys Ala Gln Arg Leu Ser Trp Leu Ile Gl - #u Gly Asp Val Val Leu           #               13501340 - #                1345                              - GAC CGC GTT GAG TCC GTT GAT GTG GAA GAC TA - #C GAT GGT TAT GTC TAT         4253                                                                          Asp Arg Val Glu Ser Val Asp Val Glu Asp Ty - #r Asp Gly Tyr Val Tyr           #              13650                                                          - GAC CTG AGC GTC GAG GAC AAC GAG AAC TTC CT - #C GTT GGC TTT GGG TTG         4301                                                                          Asp Leu Ser Val Glu Asp Asn Glu Asn Phe Le - #u Val Gly Phe Gly Leu           #          13805                                                              - GTC TAT GCT CAC AAC AGC TAC TAC GGT TAC TA - #C GGC TAT GCA AGG GCG         4349                                                                          Val Tyr Ala His Asn Ser Tyr Tyr Gly Tyr Ty - #r Gly Tyr Ala Arg Ala           #      13950                                                                  - CGC TGG TAC TGC AAG GAG TGT GCA GAG AGC GT - #A ACG GCC TGG GGA AGG         4397                                                                          Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser Va - #l Thr Ala Trp Gly Arg           #  14105                                                                      - GAG TAC ATA ACG ATG ACC ATC AAG GAG ATA GA - #G GAA AAG TAC GGC TTT         4445                                                                          Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile Gl - #u Glu Lys Tyr Gly Phe           #               14301420 - #                1425                              - AAG GTA ATC TAC AGC GAC ACC GAC GGA TTT TT - #T GCC ACA ATA CCT GGA         4493                                                                          Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe Ph - #e Ala Thr Ile Pro Gly           #              14450                                                          - GCC GAT GCT GAA ACC GTC AAA AAG AAG GCT AT - #G GAG TTC CTC AAC TAT         4541                                                                          Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Me - #t Glu Phe Leu Asn Tyr           #          14605                                                              - ATC AAC GCC AAA CTT CCG GGC GCG CTT GAG CT - #C GAG TAC GAG GGC TTC         4589                                                                          Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu Le - #u Glu Tyr Glu Gly Phe           #      14750                                                                  - TAC AAA CGC GGC TTC TTC GTC ACG AAG AAG AA - #G TAT GCG GTG ATA GAC         4637                                                                          Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Ly - #s Tyr Ala Val Ile Asp           #  14905                                                                      - GAG GAA GGC AAG ATA ACA ACG CGC GGA CTT GA - #G ATT GTG AGG CGT GAC         4685                                                                          Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu Gl - #u Ile Val Arg Arg Asp           #               15101500 - #                1505                              - TGG AGC GAG ATA GCG AAA GAG ACG CAG GCG AG - #G GTT CTT GAA GCT TTG         4733                                                                          Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Ar - #g Val Leu Glu Ala Leu           #              15250                                                          - CTA AAG GAC GGT GAC GTC GAG AAG GCC GTG AG - #G ATA GTC AAA GAA GTT         4781                                                                          Leu Lys Asp Gly Asp Val Glu Lys Ala Val Ar - #g Ile Val Lys Glu Val           #          15405                                                              - ACC GAA AAG CTG AGC AAG TAC GAG GTT CCG CC - #G GAG AAG CTG GTG ATC         4829                                                                          Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pr - #o Glu Lys Leu Val Ile           #      15550                                                                  - CAC GAG CAG ATA ACG AGG GAT TTA AAG GAC TA - #C AAG GCA ACC GGT CCC         4877                                                                          His Glu Gln Ile Thr Arg Asp Leu Lys Asp Ty - #r Lys Ala Thr Gly Pro           #  15705                                                                      - CAC GTT GCC GTT GCC AAG AGG TTG GCC GCG AG - #A GGA GTC AAA ATA CGC         4925                                                                          His Val Ala Val Ala Lys Arg Leu Ala Ala Ar - #g Gly Val Lys Ile Arg           #               15901580 - #                1585                              - CCT GGA ACG GTG ATA AGC TAC ATC GTG CTC AA - #G GGC TCT GGG AGG ATA         4973                                                                          Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Ly - #s Gly Ser Gly Arg Ile           #              16050                                                          - GGC GAC AGG GCG ATA CCG TTC GAC GAG TTC GA - #C CCG ACG AAG CAC AAG         5021                                                                          Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe As - #p Pro Thr Lys His Lys           #          16205                                                              - TAC GAC GCC GAG TAC TAC ATT GAG AAC CAG GT - #T CTC CCA GCC GTT GAG         5069                                                                          Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Va - #l Leu Pro Ala Val Glu           #      16350                                                                  - AGA ATT CTG AGA GCC TTC GGT TAC CGC AAG GA - #A GAC CTG CGC TAC CAG         5117                                                                          Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Gl - #u Asp Leu Arg Tyr Gln           #  16505                                                                      - AAG ACG AGA CAG GTT GGT TTG AGT GCT TGG CT - #G AAG CCG AAG GGA ACT         5165                                                                          Lys Thr Arg Gln Val Gly Leu Ser Ala Trp Le - #u Lys Pro Lys Gly Thr           #               16701660 - #                1665                              - TGACCTTTCC ATTTGTTTTC CAGCGGATAA CCCTTTAACT TCCCTTTCAA AA - #ACTCCCTT       5225                                                                          - TAGGGAAAGA CCATGAAGAT AGAAATCCGG CGGCGCCCGG TTAAATACGC TA - #GGATAGAA       5285                                                                          - GTGAAGCCAG ACGGCAGGGT AGTCGTCACT GCCCCGAGGG TTCAACGTTG AG - #AAGTT          5342                                                                          - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 5339 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE: Hyperthermophilic arch - #aeon                    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TG - #CCGGTTTT         60                                                                          - ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TA - #CAATAAAG        120                                                                          - CCTGGATTGT TCTACAAGAT TATGGGGGAT GAAAGATGAT CCTCGACACT GA - #CTACATAA        180                                                                          - CCGAGGATGG AAAGCCTGTC ATAAGAATTT TCAAGAAGGA AAACGGCGAG TT - #TAAGATTG        240                                                                          - AGTACGACCG GACTTTTGAA CCCTACTTCT ACGCCCTCCT GAAGGACGAT TC - #TGCCATTG        300                                                                          - AGGAAGTCAA GAAGATAACC GCCGAGAGGC ACGGGACGGT TGTAACGGTT AA - #GCGGGTTG        360                                                                          - AAAAGGTTCA GAAGAAGTTC CTCGGGAGAC CAGTTGAGGT CTGGAAACTC TA - #CTTTACTC        420                                                                          - ATCCGCAGGA CGTCCCAGCG ATAAGGGACA AGATACGAGA GCATGGAGCA GT - #TATTGACA        480                                                                          - TCTACGAGTA CGACATACCC TTCGCCAAGC GCTACCTCAT AGACAAGGGA TT - #AGTGCCAA        540                                                                          - TGGAAGGCGA CGAGGAGCTG AAAATGCTCG CCTTCGACAT TCAAACTCTC TA - #CCATGAGG        600                                                                          - GCGAGGAGTT CGCCGAGGGG CCAATCCTTA TGATAAGCTA CGCCGACGAG GA - #AGGGGCCA        660                                                                          - GGGTGATAAC TTGGAAGAAC GTGGATCTCC CCTACGTTGA CGTCGTCTCG AC - #GGAGAGGG        720                                                                          - AGATGATAAA GCGCTTCCTC CGTGTTGTGA AGGAGAAAGA CCCGGACGTT CT - #CATAACCT        780                                                                          - ACAACGGCGA CAACTTCGAC TTCGCCTATC TGAAAAAGCG CTGTGAAAAG CT - #CGGAATAA        840                                                                          - ACTTCGCCCT CGGAAGGGAT GGAAGCGAGC CGAAGATTCA GAGGATGGGC GA - #CAGGTTTG        900                                                                          - CCGTCGAAGT GAAGGGACGG ATACACTTCG ATCTCTATCC TGTGATAAGA CG - #GACGATAA        960                                                                          - ACCTGCCCAC ATACACGCTT GAGGCCGTTT ATGAAGCCGT CTTCGGTCAG CC - #GAAGGAGA       1020                                                                          - AGGTTTACGC TGAGGAAATA ACACCAGCCT GGGAAACCGG CGAGAACCTT GA - #GAGAGTCG       1080                                                                          - CCCGCTACTC GATGGAAGAT GCGAAGGTCA CATACGAGCT TGGGAAGGAG TT - #CCTTCCGA       1140                                                                          - TGGAGGCCCA GCTTTCTCGC TTAATCGGCC AGTCCCTCTG GGACGTCTCC CG - #CTCCAGCA       1200                                                                          - CTGGCAACCT CGTTGAGTGG TTCCTCCTCA GGAAGGCCCT ATGAGAGGAA TG - #AGCTGGCC       1260                                                                          - CCGAACAAGC CCGATGAAAA GGAGCTGGCC AGAAGACGGC AGAGCTATGA AG - #GAGGCTAT       1320                                                                          - GTAAAAGAGC CCGAGAGAGG GTTGTGGGAG AACATAGTGT ACCTAGATTT TA - #GATGCCAT       1380                                                                          - CCAGCCGATA CGAAGGTTGT CGTCAAGGGG AAGGGGATTA TAAACATCAG CG - #AGGTTCAG       1440                                                                          - GAAGGTGACT ATGTCCTTGG GATTGACGGC TGGCAGAGAG TTAGAAAAGT AT - #GGGAATAC       1500                                                                          - GACTACAAAG GGGAGCTTGT AAACATAAAC GGGTTAAAGT GTACGCCCAA TC - #ATAAGCTT       1560                                                                          - CCCGTTGTTA CAAAGAACGA ACGACAAACG AGAATAAGAG ACAGTCTTGC TA - #AGTCTTTC       1620                                                                          - CTTACTAAAA AAGTTAAGGG CAAGATAATA ACCACTCCCC TTTTCTATGA AA - #TAGGCAGA       1680                                                                          - GCGACAAGTG AGAATATTCC AGAAGAAGAG GTTCTCAAGG GAGAGCTCGC TG - #GCATAGTA       1740                                                                          - TTGGCTGAAG GAACGCTCTT GAGGAAAGAC GTTGAATACT TTGATTCATC CC - #GCAAAAAA       1800                                                                          - CGGAGGATTT CACACCAGTA TCGTGTTGAG ATAACCATTG GGAAAGACGA GG - #AGGAGTTT       1860                                                                          - AGGGATCGTA TCACATACAT TTTTGAGCGT TTGTTTGGGA TTACTCCAAG CA - #TCTCGGAG       1920                                                                          - AAGAAAGGAA CTAACGCAGT AACACTCAAA GTTGCGAAGA AGAATGTTTA TC - #TTAAAGTC       1980                                                                          - AAGGAAATTA TGGACAACAT AGAGTCCCTA CATGCCCCCT CGGTTCTCAG GG - #GATTCTTC       2040                                                                          - GAAGGCGACG GTTCAGTAAA CAGGTTAGGA GGAGTATTGT TGCAACCCAG GG - #TACAAAGA       2100                                                                          - ACGAGTGGAA GATTAAACTG GTGTCAAAAC TGCTCTCCCA GCTTGGTATC CC - #TCATCAAA       2160                                                                          - CGTACACGTA TCAGTATCAG GAAAATGGGA AAGATCGGAG CAGGTATATA CT - #GGAGATAA       2220                                                                          - CTGGAAAGGA CGGATTGATA CTGTTCCAAA CACTCATTGG ATTCATCAGT GA - #AAGAAAGA       2280                                                                          - ACGCTCTGCT TAATAAGGCA ATATCTCAGA GGGAAATGAA CAACTTGGAA AA - #CAATGGAT       2340                                                                          - TTTACAGGCT CAGTGAATTC AATGTCAGCA CGGAATACTA TGAGGGCAAG GT - #CTATGACT       2400                                                                          - TAACTCTTGA AGGAACTCCC TACTTTGCCA ATGGCATATT GACCCATAAC TC - #CCTGTACC       2460                                                                          - CCTCAATCAT CATCACCCAC AACGTCTCGC CGGATACGCT CAACAGAGAA GG - #ATGCAAGG       2520                                                                          - AATATGACGT TGCCCCACAG GTCGGCCACC GCTTCTGCAA GGACTTCCCA GG - #ATTTATCC       2580                                                                          - CGAGCCTGCT TGGAGACCTC CTAGAGGAGA GGCAGAAGAT AAAGAAGAAG AT - #GAAGGCCA       2640                                                                          - CGATTGACCC GATCGAGAGG AAGCTCCTCG ATTACAGGCA GAGGGCCATC AA - #GATCCTGG       2700                                                                          - CAAACAGCAT CCTACCCGAG GAATGGCTTC CAGTCCTCGA GGAAGGGGAG GT - #TCACTTCG       2760                                                                          - TCAGGATTGG AGAGCTCATA GACCGGATGA TGGAGGAAAA TGCTGGGAAA GT - #AAAGAGAG       2820                                                                          - AGGGCGAGAC GGAAGTGCTT GAGGTCAGTG GGCTTGAAGT CCCGTCCTTT AA - #CAGGAGAA       2880                                                                          - CTAACAAGGC CGAGCTCAAG AGAGTAAAGG CCCTGATTAG GCACGATTAT TC - #TGGCAAGG       2940                                                                          - TCTACACCAT CAGACTGAAG TCGGGGAGGA GAATAAAGAT AACCTCTGGC CA - #CAGCCTCT       3000                                                                          - TCTCTGTGAG AAACGGGGAG CTCGTTGAAG TTACGGGCGA TGAACTAAAG CC - #AGGTGACC       3060                                                                          - TCGTTGCAGT CCCGCGGAGA TTGGAGCTTC CTGAGAGAAA CCACGTGCTG AA - #CCTCGTTG       3120                                                                          - AACTGCTCCT TGGAACGCCA GAAGAAGAAA CTTTGGACAT CGTCATGACG AT - #CCCAGTCA       3180                                                                          - AGGGTAAGAA GAACTTCTTT AAAGGGATGC TCAGGACTTT GCGCTGGATT TT - #CGGAGAGG       3240                                                                          - AAAAGAGGCC CAGAACCGCG AGACGCTATC TCAGGCACCT TGAGGATCTG GG - #CTATGTCC       3300                                                                          - GGCTTAAGAA GATCGGCTAC GAAGTCCTCG ACTGGGACTC ACTTAAGAAC TA - #CAGAAGGC       3360                                                                          - TCTACGAGGC GCTTGTCGAG AACGTCAGAT ACAACGGCAA CAAGAGGGAG TA - #CCTCGTTG       3420                                                                          - AATTCAATTC CATCCGGGAT GCAGTTGGCA TAATGCCCCT AAAAGAGCTG AA - #GGAGTGGA       3480                                                                          - AGATCGGCAC GCTGAACGGC TTCAGAATGA GAAAGCTCAT TGAAGTGGAC GA - #GTCGTTAG       3540                                                                          - CAAAGCTCCT CGGCTACTAC GTGAGCGAGG GCTATGCAAG AAAGCAGAGG AA - #TCCCAAAA       3600                                                                          - ACGGCTGGAG CTACAGCGTG AAGCTCTACA ACGAAGACCC TGAAGTGCTG GA - #CGATATGG       3660                                                                          - AGAGACTCGC CAGCAGGTTT TTCGGGAAGG TGAGGCGGGG CAGGAACTAC GT - #TGAGATAC       3720                                                                          - CGAAGAAGAT CGGCTACCTG CTCTTTGAGA ACATGTGCGG TGTCCTAGCG GA - #GAACAAGA       3780                                                                          - GGATTCCCGA TGGCGTCTTC ACGTCCCCGA AAGGGGTTCG GCTGGCCTTC CT - #TGAGGGGT       3840                                                                          - ACTCATCGGC GATGGCGACG TCCACCGAAC AAGAGACTCA GGCTCTCAAC GA - #AAAGCGAG       3900                                                                          - CTTTAGCGAA CCAGCTCGTC CTCCTCTTGA ACTCGGTGGG GGTCTCTGCT GT - #AAAACTTG       3960                                                                          - GGCACGACAG CGGCGTTTAC AGGGTCTATA TAAACGAGGA GCTCCCGTTC GT - #AAAGCTGG       4020                                                                          - ACAAGAAAAA GAACGCCTAC TACTCACACG TGATCCCCAA GGAAGTCCTG AG - #CGAGGTCT       4080                                                                          - TTGGGAAGGT TTTCCAGAAA AACGTCAGTC CTCAGACCTT CAGGAAGATG GT - #CGAGGACG       4140                                                                          - GAAGACTCGA TCCCGAAAAG GCCCAGAGGC TCTCCTGGCT CATTGAGGGG GA - #CGTAGTGC       4200                                                                          - TCGACCGCGT TGAGTCCGTT GATGTGGAAG ACTACGATGG TTATGTCTAT GA - #CCTGAGCG       4260                                                                          - TCGAGGACAA CGAGAACTTC CTCGTTGGCT TTGGGTTGGT CTATGCTCAC AA - #CAGCTACT       4320                                                                          - ACGGTTACTA CGGCTATGCA AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GA - #GAGCGTAA       4380                                                                          - CGGCCTGGGG AAGGGAGTAC ATAACGATGA CCATCAAGGA GATAGAGGAA AA - #GTACGGCT       4440                                                                          - TTAAGGTAAT CTACAGCGAC ACCGACGGAT TTTTTGCCAC AATACCTGGA GC - #CGATGCTG       4500                                                                          - AAACCGTCAA AAAGAAGGCT ATGGAGTTCC TCAACTATAT CAACGCCAAA CT - #TCCGGGCG       4560                                                                          - CGCTTGAGCT CGAGTACGAG GGCTTCTACA AACGCGGCTT CTTCGTCACG AA - #GAAGAAGT       4620                                                                          - ATGCGGTGAT AGACGAGGAA GGCAAGATAA CAACGCGCGG ACTTGAGATT GT - #GAGGCGTG       4680                                                                          - ACTGGAGCGA GATAGCGAAA GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CT - #AAAGGACG       4740                                                                          - GTGACGTCGA GAAGGCCGTG AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AG - #CAAGTACG       4800                                                                          - AGGTTCCGCC GGAGAAGCTG GTGATCCACG AGCAGATAAC GAGGGATTTA AA - #GGACTACA       4860                                                                          - AGGCAACCGG TCCCCACGTT GCCGTTGCCA AGAGGTTGGC CGCGAGAGGA GT - #CAAAATAC       4920                                                                          - GCCCTGGAAC GGTGATAAGC TACATCGTGC TCAAGGGCTC TGGGAGGATA GG - #CGACAGGG       4980                                                                          - CGATACCGTT CGACGAGTTC GACCCGACGA AGCACAAGTA CGATGCCGAG TA - #CTACATTG       5040                                                                          - AGAACCAGGT TCTCCCAGCC GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CG - #CAAGGAAG       5100                                                                          - ACCTGCGCTA CCAGAAGACG AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CC - #GAAGGGAA       5160                                                                          - CTTGACCTTT CCATTTGTTT TCCAGCGGAT AACCCTTTAA CTTCCCTTTC AA - #AAACTCCC       5220                                                                          - TTTAGGGAAA GACCATGAAG ATAGAAATCC GGCGGCGCCC GGTTAAATAC GC - #TAGGATAG       5280                                                                          - AAGTGAAGCC AGACGGCAGG GTAGTCGTCA CTGCCCCGAG GGTTCAACGT TG - #AGAAGTT        5339                                                                          - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24AAGG CGAC                                                  - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24CCGA GCTT                                                  - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 324 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - GGATTAGTGC CAATGGAAGG CGACGAGGAG CTGAAAATGC TCGCCTTCGA CA - #TTCAAACT         60                                                                          - CTCTACCATG AGGGCGAGGA GTTCGCCGAG GGGCCAATCC TTATGATAAG CT - #ACGCCGAC        120                                                                          - GAGGAAGGGG CCAGGGTGAT AACTTGGAAG AACGTGGATC TCCCCTACGT TG - #ACGTCGTC        180                                                                          - TCGACGGAGA GGGAGATGAT AAAGCGCTTC CTCCGTGTTG TGAAGGAGAA AG - #ACCCGGAC        240                                                                          - GTTCTCATAA CCTACAACGG CGACAACTTC GACTTCGCCT ATCTGAAAAA GC - #GCTGTGAA        300                                                                          #               324TCGC CCTC                                                  - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 108 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gly Leu Val Pro Met Glu Gly Asp Glu Glu Le - #u Lys Met Leu Ala Phe         #15                                                                           - Asp Ile Gln Thr Leu Tyr His Glu Gly Glu Gl - #u Phe Ala Glu Gly Pro         #             30                                                              - Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu Gl - #y Ala Arg Val Ile Thr         #         45                                                                  - Trp Lys Asn Val Asp Leu Pro Tyr Val Asp Va - #l Val Ser Thr Glu Arg         #     60                                                                      - Glu Met Ile Lys Arg Phe Leu Arg Val Val Ly - #s Glu Lys Asp Pro Asp         # 80                                                                          - Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe As - #p Phe Ala Tyr Leu Lys         #                 95                                                          - Lys Arg Cys Glu Lys Leu Gly Ile Asn Phe Al - #a Leu                         #           105                                                               - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #  42              CAAA CAGCTACTAC GGTTACTACG GC                              - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: other nucleic acid                                  #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #          32      CAAC GTTGAACCCT CG                                         - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                 46ATT TTAGATCCCT GTACCCCTCA ATCATC                          - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #  42              TAGC TGTTTGCCAG GATCTTGATG GC                              - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #         33       CTGA CTACATAACC GAG                                        - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                 46GGG ATCTAAAATC TAGGTACACT ATGTTC                          __________________________________________________________________________

What we claim is:
 1. A thermostable DNA polymerase which is a strainKOD1 hyperthermophilic archaeon enzyme.
 2. The thermostable DNApolymerase of claim 1, which has an amino acid sequence of SEQ ID No. 1.3. The thermostable DNA polymerase of claim 1, which has the followingphysical and chemical properties;Action: catalyzing the extensionreaction of nucleotide sequence that is complementary to a templatenucleotide sequence, using nucleotide triphosphate as substrate andhaving a 3'-5' exonuclease activity, DNA extension rate: at least 30bases/second Optimum pH: 6.5-7.5 (at 75° C.) Optimum temperature: 75° C.Molecular weight: about 88-90 Kda Amino acid sequence: SEQ ID No.
 1. 4.The DNA polymerase of claim 1, which is a protein.